Metabolism of sulphur

Abstract

Of the enzymes which act on the S-containing amino-acids in the tissue, four have been described previously: (a) cytochrome oxidase, which oxidizes cysteine to cystine, (b) a cysteine oxidase, described by Pirie [1934], which gives rise to inorganic sulphate, (c) a sulphinic acid oxidase, reported by Medes [1939], also producing inorganic sulphate, and (d) a cysteine oxidase, reported by Bernheim & Bernheim [1939] and Medes [1939], which oxidizes cysteine to cysteic acid. Medes's results also suggested the direct oxidation of cystine, accompanied by decarboxylation. At that time it was not clear whether the action on cystine and on cysteine was due to the same or to different enzymes, or through what paths the oxidations proceeded. The series ofobservations reported here are attempts to elucidate some of these questions. STUDIES WITH LIVER SLICES A preliminary study was made of the action of liver slices on the S-containing aminoacids in order to identify the several end-products. Such a study should serve as a guide in determining whether all enzymes catalysing the oxidation of these substrates had been isolated. The slices were incubated in a constant temperature bath at 380 in phosphate or bicarbonate buffer at pH 7-6 with cysteine, cystine, methionine and cysteinesulphinic acid as substrates. The bicarbonate buffer was prepared as recommended by Krebs [1932] with substitution of MgC12 for MgSO4 as Pirie [1934] suggested. When phosphate buffer was employed, its final concentration was 0*05 M. Volumetric analyses for the various end-products were carried out after 2 hr. incubation with 02 bubbling through the phosphate buffer and an 02-CO2 mixture led through the bicarbonate buffer. Methods of quantitative analyses. Cysteine and cystine were determined by the method of Shinohara & Padis [1935] with occasional checks by the Sullivan method. For the determination of methionine added to tissue extract the precision method of Kolb & Toennies [1940] was adapted. An aliquot part of the brei containing about 0*1 m.mol. of methionine is placed in a 50 ml. 'centrifuge tube and is diluted with 20 % trichloroacetic acid and H20 to 50 ml. so that the resulting solution is about 0-002 M in methionine and contains approximately 4 % trichloroacetic acid. All volumes are measured accurately. This mixture is stirred vigorously, allowed to stand for 5 min. to ensure adequate flocculation of the protein, and centrifuged. A 40 ml. aliquot part of the supernatant liquid is placed in a 100 ml. volumetric flask containing 36 ml. of 5 M HC1104. To this are added 10 ml. of 2 M HC104 containing 0-06 M H2°02,

Sulphate formation from dimethylthetin in rat liver

“…At lower levels the percentage oxidation was higher, 6 % at a level of 2 mg. and 10 % at a level of 1 mg. This is of the same order as that obtained with equivalent amounts of cysteine, both in the present experiments and in those of Pirie (1934a) and Medes (1939).…”

“…Pirie (1934a, b) observed the oxidation to sulphate of cysteine, cystine, glutathione, methionine and a number of other compounds by slices of rat tissues, of which liver and kidney were the most active. Medes (1939) and Medes & Floyd (1942) confirmed these findings and extended their experiments to liver breis and extracts. Sulphate was also produced by tissue slices and extracts without the addition of sulphur-containing compounds to the medium.…”

“…A period of 1 hr. in ice has been suggested by Cope (1931) and by Medes & Floyd (1942) as being adequate for quantitative precipitation of benzidine sulphate under these conditions. Pirie (1934a) suggested a minimum period of 2 hr.…”

“…Sulphate was also produced by tissue slices and extracts without the addition of sulphur-containing compounds to the medium. Medes (1939) gave average values for these tissue sulphate 'blanks' corresponding to 28-7 pg. sulphate-S/100 mg. dry wt.…”

The biosynthesis of penicillin. 2. The incorporation of cystine into penicillin

“…It is known (cf. Arnstein & Grant, 1954) that cystine is oxidized to carbon dioxide during the fermentation and it is likely that in this reaction the sulphur of cystine is converted into sulphate, possibly by a pathway similar to that known to occur in animal tissue (Medes & Floyd, 1942;cf. Kearney & Singer, 1953).…”